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Sample GSM6592535

Query DataSets for GSM6592535

Status

Public on Sep 30, 2023

Title

Metabolic cycle 2, biol rep 1

Sample type

SRA

Source name

yeast cell

Organism

Saccharomyces cerevisiae

Characteristics

strain: CEN.PK
cell type: yeast cell
genotype: MATa URA3 TRP1 LEU2 HIS3 MAL2-8C SUC2
treatment: 32 min from the start of oxidative phase

Treatment protocol

Heat shock was conducted at 37°C for 20 min. To obtain cells experiencing diauxic shift, the glucose concentration of a culture was determined by Quantichrom glucose assay kit (Bioassay systems) following the manufacturer’s instructions and the culture with diauxic shift was harvested at 2 h after glucose depletion. For DNA damage, a culture was treated with 5 µg/ml 4-nitroquinoline 1-oxide (Sigma) for 2 h. The period of metabolic oscillation was approximately 3.2 h. Cells were harvested at 16, 32, 48, and 60 min from the start of the oxidative phase of each cycle in three consecutive oscillations.

Growth protocol

Yeast strain BY4741 was grown in YPD medium. An overnight culture was inoculated into 600 ml of YPD with a starting OD600 of 0.2. When the culture was grown to mid-log phase, it was dispensed by 100 ml and each was subjected to either control (30°C for 20 min or 2 h) or one of the three stress conditions (diauxic shift, DNA damage, heat shock). For metabolic cycles, yeast strain CEN.PK was cultivated as described elsewhere (Nocetti and Whitehouse, Genes & Dev., 2016).

Extracted molecule

total RNA

Extraction protocol

The total RNA from each sample was extracted by hot phenol method.
An oligo(dT) primer containing a VN-anker, a uridine, the sequence of the sequencing adapter, and biotin (/5BiosG/CAGACGTGTGCTCTTCCGATCTTTTTTTTrUTT TTTTTTVN) was attached to streptavidin-coated magnetic beads (Invitrogen, M280). Total RNA was incubated for 5 min at 65°C, followed by incubation with the coated magnetic beads for 10 min at 45°C. First strand synthesis was carried out using SuperScript III reverse transcriptase (Invitrogen) at 50°C. The enzyme was inactivated by incubation for 15 min at 70°C. Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen). To exchange the buffers and remove enzymes from the previous step, we treated the samples with Pronase (Roche). We introduced a nick at the position of the uridine with RNase HII (New England Biolabs). Then, nick translation was carried out with DNA polymerase I (New England Biolabs) and dNTPs for 8 min at 8°C. The reaction was stopped by the addition of EDTA. At the new position of the nick, we created a blunt end using T7 exonuclease, mung bean nuclease, and Klenow enzyme (New England Biolabs). Previously annealed Illumina TruSeq sequencing adapters (Ad1, 59-AATGATACGGCGACCACCGAGATC TACACTCTTTCCCTACACGACGCTCTTCCGATCT-39;and Ad2, 59-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT AGATCTCGGTGGTCGCCGTAT-39) were then blunt-end-ligated. The magnetic beads were suspended in 39 mLofH2O. Five microliters of beads was used in a PCR reaction. Ten to 13 cycles of PCR were carried out with Phusion polymerase (New England Biolabs), a forward primer (59-AATGATACGGCGACCACCGA GATC-3), and one of the Illumina TruSeq barcode reverse primers. For preparation of the library, six PCR reactions were performed and run on 8% TBE gels (Invitrogen), and the smear between 160 bp and 220 bp was cut out, gel-extracted, ethanol-precipitated, analyzed by Bioanalyzer (Agilent), and sequenced on Illumina HiSeq machines.
3'-seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

ymc2.F.bedgraph
ymc2.R.bedgraph

Data processing

Low-quality ends were trimmed by trimmomatic (version: 0.39; average quality per base < 15, 4 bp window). 3'-adapters were removed by cutadapt (version: 2.3)
Reads with at least two terminal adenylates were selected. The number of terminal adenylates were recorded and appended to read ID. All terminal adenylates were trimmed off from each read, and remaining sequence was mapped to yeast genome (version: 64.1.1) using hisat2 (version: 2.1.0).
Only reads with high mapping quality (mapq > 30) were kept. Only those reads whose number of terminal adenylates (labels in their IDs) exceeds the number of consecutive adenylates in adjacent genomic regions.
Bedgraph files containing genome coverage for each sample were generated by bedtools (genomecov function; version: v2.26.0)
Assembly: sacCer3
Supplementary files format and content: F.bedgraph (Bedgraph file of genome coverage on plus strand; all three biological replicates combined)
Supplementary files format and content: R.bedgraph (Bedgraph file of genome coverage on minus strand; all three biological replicates combined)
The bedgraph files are the read coverage of 3’-seq data. They represent the location of cleavage and polyadenylation sites.

Submission date

Sep 19, 2022

Last update date

Sep 30, 2023

Contact name

Christine Mayr

E-mail(s)

mayrc@mskcc.org

Organization name

MSKCC

Department

Cancer Biology and Genetics